miR-148a对肝癌细胞株侵袭和迁移的抑制作用及机制

摘要

Background and purpose: Primary liver cancer is the malignant tumor of liver cells or intrahepatic bile duct epithelium with familiar metastasis and postsurgical recurrence. The purpose of this study was to investigate the effects of miR-148a on the invasion and migration of hepatocellular carcinoma cells and the underlying mechanisms. Methods: The supernatant containing LV-miR-148a lentivirus particles was used to infect SMMC-7721 cells. The expression of miR-148a was determined by RT-PCR. Wound healing assay and transwell assay were performed to detect the effects of miR-148a on the invasion of hepatocellular carcinoma cells. Gelatin zymography assay was used to detect the effects of miR-148a on the enzyme activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The expression of MMP-2, MMP-9, E-cadherin and vimentin proteins was detected by Western blot assay. Results:RT-PCR showed the expression of miR-148a was upregulated in the infected SMMC-7721 cells. Transwell assay and wound healing assay showed ectopic expression of miR-148a suppressed cell migration and invasion abilities. miR-148a overexpression led to the decrease of the enzyme activities of MMP-2 and MMP-9 (P<0.05). Western blot assay showed that the protein expression of MMP-2, MMP-9 and vimentin proteins was signiifcantly decreased, the expression of E-cadherin had no changes. Conclusion:miR-148a is able to inhibit the migration and invasion of human SMMC-7721 cells in vitro, and the possible mechanisms may be related to decrease the enzyme activities of the MMP-2 and MMP-9 and the down regulation expression of MMP-2, MMP-9 and vimentin.%背景与目的：原发性肝癌是肝细胞或肝内胆管上皮发生的恶性肿瘤,其复发和转移十分常见。本实验以慢病毒介导miR-148a,感染人肝癌SMMC-7721细胞,观察其对SMMC-7721细胞侵袭和迁移能力的影响。方法：选用SMMC-7721肝癌细胞株，进行miR-148a慢病毒载体的构建，筛选稳定表达miR-148a肝癌细胞株。RT-PCR检测细胞中miR-148a的表达水平。划痕实验及Transwell侵袭实验检测细胞侵袭迁移能力。采用明胶酶谱法测定MMP-2、MMP-9的活性。蛋白质印迹法(Western blot)检测MMP-2、MMP-9和EMT相关蛋白(E-cadherin、vimentin)的表达情况。结果：RT-PCR结果显示，和对照组相比，LV-miR-148a肝癌细胞感染组表达miR-148a明显增高，体外侵袭实验细胞划痕实验显示过表达miR-148a后SMMC-7721的侵袭和迁移能力明显下降。明胶酶谱法结果显示过表达miR-148a的肝癌细胞，MMP-2和MMP-9降解明胶的能力下降(P<0.05)。过表达miR-148a同时能降低肝癌细胞SMMC-7721中vimentin、MMP-2和MMP-9蛋白的表达，但并未影响E-cadherin的表达。结论：miR-148a在体外能抑制肝癌细胞SMMC-7721的侵袭和迁移，其机制可能与下调MMP-2、MMP-9和vimentin的表达水平有关。